Monoclonal antibodies against osteopontin

ABSTRACT

The present invention relates to reagents and methods for the detection of osteopontin fragments and distinguishing them from each other and from the full-length osteopontin protein. The present invention also relates to assays for the determination of the presence of osteopontin fragments in samples obtained from subjects and, further, the correlation of osteopontin fragment levels fragment levels with disease detection, progression and prognosis.

The Applicants acknowledge the National Cancer Institute of the NationalInstitutes of Health for financial support of this work under Grant No.CA129849. The Federal Government has a nonexclusive, nontransferable,irrevocable license in this invention on behalf of the United States.[37 CFR 401.14(b)].

BACKGROUND

Osteopontin (OPN) is a secreted phosphoprotein that is associated withcancer, cardiovascular disease, renal injury and inflammation. One ofthe first descriptions of osteopontin was as a secreted phosphoprotein(spp 1) found at elevated levels circulating in the serum of cancerpatients. Since that time, numerous studies have shown upregulatedexpression of osteopontin in various human cancers. Not limited tocancer, osteopontin expression is associated with various tissue injuryand human diseases. Several clinical studies have been performed toassess the potential of osteopontin to serve as a clinical marker fordisease progression or prognosis in human breast cancer. The results ofthese studies show that there is a significant overlap of osteopontinwith other variables associated with patient outcome including highhistological grade, c-ErbB3 and p53 (Rudland, et al., Cancer Res,62(12):3417-3427, 2002). Significantly, these breast cancer patientswere studied for 14-20 years of follows up and, while thelow-osteopontin group had a median survival of >228 months, thehigh-osteopontin group had a median survival of 68 months, suggestingpredictive value of osteopontin levels in long-term patient outcome.Other studies suggest that, in addition to breast cancer, elevatedosteopontin in the serum is associated with prostate and lung cancer(Fedarko, et al., Clin Cancer Res 7(12):4060-4066, 2001).

There are several characteristics of osteopontin that make it a valuablecandidate as a biomarker for disease. The protein is secreted in bodyfluids and can be found in, e.g., plasma and serum, human milk andurine. There are available ELISA-based assays to test for osteopontinprotein levels. However, multiple modified fragments of osteopontinexist that represent proteolytically cleaved fragments of the parentmolecule. These cleaved fragments are distinct functionally (Gao, Y. A.,et al., Matix Biol, 23(7):457-466, 2004; Senger, D. R., et al., Ann NYAcd Sci, 760:83-100, 1995; Senger, D. R., et al., Am J Pathol,149(1):293-305, 1996; Senger D. R., et al., Biochem Biophys Acta,1314(1-2):13-24, 1996; Bayless, K. J., et al., J Biol Chem,276(16):13483-13489, 2001; Green, P. M., et al., FEBS Lett,503(1):75-79, 2001; Smith, L. L., et al., Exp Cell Res, 242:351-360,1998; Yokosaki, Y., et al., Matrix Biol, 24(6):418-427, 2005), theyoccur in vivo and are generated through the catalytic activity ofproteases such as thrombin and the matrix metalloproteinases that areknown to be associated with tumor progression. Indeed, the question ofwhether these osteopontin fragments wound provide a more accurateassessment of clinical tumor burden, progression or patient outcome isan important issue that has not been addressed. Part of the limitationis that there are not reagents that will specifically detect osteopontinfragments and distinguish them from each other and the full-lengthprotein. Thus far only SDS-PAGE analysis followed by immunoblotting withanti-osteopontin antibodies can show fragment presence in clinicalsamples. However, even this method has significant drawbacks sinceantibodies for specific OPN fragments may not exist. For example, arecent report showed that results of commercially available ELISA assaysfor osteopontin in neck, head and cervix cancer patents are dependentupon the assay kit used (Vordermark, et al, “Plasma osteopontin levelsin patients with head and neck cancer and cervix cancer are criticallydependent on the choice of ELISA system” BMC Cancer, 6(1):207, 2006).The authors did not consider that the differences between the assays maybe due to the detection (or lack of detection) of the specificosteopontin fragments that correlate with disease presence, progressionand prognosis.

Therefore, what is needed are reagents and methods that willspecifically detect osteopontin fragments and distinguish them from eachother and from the full-length protein.

SUMMARY OF THE INVENTION

The present invention relates to reagents and methods for the detectionof osteopontin fragments and distinguishing them from each other andfrom the full-length osteopontin protein. The present invention alsorelates to assays for the determination of the presence of osteopontinfragments in samples obtained from subjects and, further, thecorrelation of osteopontin fragment levels with disease detection,progression and prognosis.

In one embodiment, the present invention relates to an assay designed,for example, to detect osteopontin fragments in a sample. The sample maybe from a subject or may be a freshly obtained or a stored sample (e.g.,a frozen sample). The sample may be, for example, serum, plasma, blood,breast milk, sputum, semen, wound secretions, tears, mucous or any otherbodily fluid or secretion known or suspected of comprising osteopontin.The sample may also be a tissue or tissue homogenate. The subject may beany living organism known to produce osteopontin and/or osteopontinfragments or suspected of producing osteopontin and/or osteopontinfragments. For example, the subject may be any mammal including humans.Additionally, the sample may be derived from in vitro sources such assynthesized peptides and peptide fragments or peptides and fragmentsmade recombinantly. Such synthetic or recombinantly made peptides andfragments may be used to, for example, calibrate an assay, serve aspositive and/or negative controls or test the efficacy of new antibodiesfor the ability to recognize and bind osteopontin and osteopontinfragments.

The present invention is not limited to any particular assay format forthe detection of osteopontin fragments. Any assay capable of detectingosteopontin fragments and distinguishing them from full-lengthosteopontin is within the scope of the present invention. By way ofexample, in one embodiment, the present invention contemplates an assaycomprising capturing an osteopontin fragment with a capture antibodywhich binds to a first epitope of the osteopontin fragment, detectingthe captured osteopontin fragment with an antibody that bindsspecifically to a second epitope of the osteopontin fragment and,determining that the peptide is a fragment by detecting a lack ofbinding by a determining antibody that binds specifically to a thirdepitope of osteopontin, as compared to a suitable control. The use ofthree antibodies, as opposed to an assay using just two antibodies,increases accuracy and decreases erroneous results by, for example,decreasing the occurrence of false positives.

In another embodiment of the present invention, an assay is contemplatedcomprising capturing an osteopontin fragment with a capture antibodywhich binds specifically to either the N-terminus or C-terminus of theosteopontin fragment and determining that the peptide is a fragment bydetecting a lack of binding by a determining antibody that bindsspecifically to the C-terminus of the osteopontin protein if an antibodyspecific for the N-terminus was used as the capture antibody or bydetecting a lack of binding by a determining antibody that bindsspecifically to the N-terminus of the osteopontin protein if an antibodyspecific for the C-terminus was used as the capture antibody, ascompared to a suitable control.

Furthermore, the present invention relates to antibodies withspecificity to osteopontin fragments. Although the present invention isnot limited to any specific antibody or antibodies, the presentinvention relates, for example, to antibodies designated 2C5, 2H9, 2F10,2E11 and 1F11, which have been shown to have specific reactivity withthe N-terminal fragment of OPN or the C-terminal fragment of OPN, asdescribed in detail, infra.

Other embodiments of the present invention will become evident to onepracticed in the art based on the teachings as given in the DetailedDescription of the Invention and the Exemplification sections of thisdocument.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the antibody titration of various mouse bleeds. 1285preImm: Blood serum from OPN-null mouse strain 1285 before the mouse wasimmunized with h-FL OPN. 1284 1^(st) bld (H+L): Blood serum collectedfrom mouse strain 1284 after immunizing five times with h-FL OPN andwith the second antibody HRP-conjugated goat anti-mouse IgG+IgM heavyand light chain used in this assay. 1284 1^(st) bld (Fc): Same as 12841^(st) bld except the second antibody was HRP-conjugated goat anti-mouseIgG specific for the antibody's Fc portion.

FIG. 2 shows an SDS-PAGE analysis of purified MAbs against OPN usingantibodies 1F11 and 2F10 (2A) and 2E11, 2C5 and 2H9 (2B).

FIG. 3 shows normalized titration of (3A) hybridoma supernatant, (3B)ascite fluid and (3C) biotinylated MAbs. The assays were preformed usingan ELISA assay with 10-fold serially diluted antibodies. The antibodytiters were normalized based on their concentrations using the followingformula: Titer=A405 nm/Starting Antibody Concentration (mg/ml).

FIG. 4 shows specificity differentiation testing of the anti-OPNmonoclonal antibodies using an ELISA. Error bar+1 standard error of mean(SEM).

FIG. 5 shows cross-reactivity of MAbs against human and mouse OPN. Thesupernatants from the five monoclonal hybridoma (2E11, 2C5 2H9, 1F11 and2F10) were probed against h-FL OPN and m-FL OPN in (5A) ELISA and inWestern blot (5B=human probe and 5C=murine probe).

FIG. 6 shows the determination of epitope specifics on the anti-OPN MAbsby (6A) ELISA and (6B & 6C) Western blot. Antibodies were probed againstN-terminal and C-terminal fragments of OPN.

FIG. 7 shows a comparison of epitopes recognized by the MAbs of thepresent invention using competition ELISA against (7A) C-terminal humanFL OPN and (7B) N terminal human OPN.

FIG. 8 shows a Western blot analysis of anti-OPN MAbs against native OPNin rat kidney and skull lysate.

FIG. 9 shows an immunohistochemical analysis of OPN in mouse bone by(9A) a control antibody or (9B & 9C) biotinylated 1F11. (9A) is at 100×,(9B) is at 50× and (9C) is at 200× magnification. (9C) is a high powerview of a portion of the field shown in (9B).

FIG. 10 shows the characterization of the monoclonal antibodies of thepresent invention for neutralization function against OPN-mediated celladhesion. (10A) shows a dose response curve of OPN to the non-invasivebreast cancer cell line MCF7, mouse embryonic fibroblast cell line (MEF)and mouse mammary tumor cell line (PyVT). (10B) shows MAb inhibition ofcell adhesion to OPN.

FIG. 11 shows an OPN standard curve generated with a sandwich ELISA. Asandwich ELISA was performed with 2F10 as the capture antibody andbiotinylated 1F11 as the detection antibody. A dose curve of humanrecombinant osteopontin was used. The low range of the samples isdepicted here (0.9 ng/ml to 14 pg/ml) to show assay sensitivity. Therewas a high correlation coefficient with R²=0.99.

FIG. 12 shows a quantitative analysis of OPN in human papillarycarcinoma (PTC) and kidney transplant patients using a sandwich ELISA.(12A) shows the mean concentration for each group. (12B) shows thedistribution of individual absorbency values for each group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to reagents and methods for the detectionof osteopontin fragments and distinguishing them from each other andfrom the full-length osteopontin protein. The present invention alsorelates to assays for the determination of the presence of osteopontinfragments in samples obtained from subjects and, further, thecorrelation of osteopontin fragment levels fragment levels with diseasedetection, progression and prognosis.

As defined herein, the term “fragments” refer to any peptide having thefull-length osteopontin sequence less one or more amino acids including,but not limited to, splice variants, mutations, deletions,substitutions, etc.

As defined herein, the terms “N-terminal” and “N-terminus” of theosteopontin protein fragment refers to, for example, approximately aminoacids 1-166 of the osteopontin protein when cleaved by matrixmetalloproteinases, or portion thereof. Also, as defined herein, theterms “N-terminal” and “N-terminus” of the osteopontin protein fragmentrefers to, for example, approximately amino acids 1-168 of theosteopontin protein when cleaved by thrombin, or portion thereof. Thosepracticed in the art will recognize that other proteases will cleaveosteopontin at different cleavage sites thereby resulting in“N-terminal” and “N-terminus” fragments of varying sizes. Additionally,osteopontin fragments may be made synthetically and may includeN-terminal fragments of various sizes.

As defined herein, the terms “C-terminal” and “C-terminus” of theosteopontin protein fragment refers to, for example, approximately aminoacids 167-314 of the osteopontin protein when cleaved by matrixmetalloproteinases, or portion thereof. Also, as defined herein, theterms “C-terminal” and “C-terminus” of the osteopontin protein fragmentrefers to, for example, approximately amino acids 169-314 of theosteopontin protein when cleaved by thrombin, or portion thereof. Thosepracticed in the art will recognize that other proteases will cleaveosteopontin at different cleavage sites thereby resulting in“C-terminal” and “C-terminus” fragments of varying sizes. Additionally,osteopontin fragments may be made synthetically and may includeC-terminal fragments of various sizes.

As defined herein, the term “capture antibody” refers to an antibodythat is capable of being removably or irremovable attached to a solidsurface and is capable of binding to a target epitope on a targetmolecule (e.g., a peptide and, more preferably, an osteopontin peptideor fragment thereof).

As defined herein, the term “detecting antibody” refers to an antibodythat is capable of binding to a target epitope on the target moleculepreferably after the target molecule has been captured by a capturingantibody. Also, preferably, the detecting antibody is specific for anepitope that is different than the epitope recognized by the captureantibody.

As defined herein, the term “determining antibody” refers to an antibodythat is capable of binding to an epitope on the same target molecule asthe capturing and detecting antibodies, wherein said epitope is notrecognized by either the capture or detecting antibody. In oneembodiment of the invention, the target molecule is an osteopontinfragment and the target molecule for the determining antibody is locatedon the portion of the osteopontin fragment that has not been captured bythe capture molecule.

As defined herein, the term “binds specifically” or similar terms, whenused in the context of an antibody binding a target epitope, refers tothe antibody having specificity for the target epitope (as opposed toother epitopes). The specificity need not be 100%. In one embodiment,the specificity is about 75% or greater (i.e., 75% specificity for theepitope). This means that about 75% of the antibodies that bind to anepitope will bind to the target epitope and about 25% of the antibodieswill bind non-specifically. In another embodiment, the specificity isabout 90% or greater.

In one embodiment, the present invention relates to an assay designed,for example, to detect osteopontin fragments in a sample. The sample maybe from a subject. The sample may comprise, for example, serum, plasma,blood, breast milk, sputum, semen, wound secretions, tears, urine,mucous or any other bodily fluid or secretion. Additionally, the samplemay be a tissue sample. The subject may be any living organism known orsuspected of producing osteopontin and/or osteopontin fragments. Forexample, the subject may be any mammal including humans. Additionally,the sample may be derived from in vitro sources such as synthesizedpeptides and peptide fragments or peptides and fragments maderecombinantly. Such synthetic or recombinantly made peptides andfragments may be used to, for example, calibrate an assay, serve aspositive and/or negative controls or test the efficacy of new antibodiesfor the ability to recognize and bind osteopontin and osteopontinfragments.

The present invention is not limited to any particular assay format forthe detection of osteopontin fragments. Any assay capable of detectingosteopontin fragments and distinguishing them from full-lengthosteopontin is within the scope of the present invention. By way ofexample, in one embodiment, the present invention contemplates an assaycomprising capturing an osteopontin fragment with a capture antibodywhich binds to a first epitope of the osteopontin fragment, detectingthe captured osteopontin fragment with an antibody that bindsspecifically to a second epitope of the osteopontin fragment and,determining that the peptide is a fragment by detecting a lack ofbinding by a determining antibody that binds specifically to a thirdepitope of the osteopontin fragment.

The present invention is not limited by the antibodies used so long asthey meet the criteria of binding specifically to different epitopes ofthe osteopontin protein, as defined above. For example, in oneembodiment, the capture and detecting antibodies bind to differentepitopes on the N-terminus of the osteopontin protein and thedetermining antibody binds specifically to an epitope on the C-terminusof the osteopontin protein. In another embodiment, the capture anddetecting antibodies bind to different epitopes on the C-terminus of theosteopontin protein and the determining antibody binds specifically toan epitope on the N-terminus of the osteopontin protein. In anotherembodiment, the capture and detecting antibodies bind to the same orsimilar epitope but are used in sub-saturating concentrations so thatthe epitope is not saturated by the capture antibody before theintroduction of the detecting antibody. In one embodiment, theantibodies are monoclonal, polyclonal or a mixture of monoclonal andpolyclonal antibodies.

In a preferred embodiment, the antibodies of the present invention aremonoclonal antibodies designated were submitted to the ATCC (Manassas,Va.) on Nov. 2, 2010 for deposit as required under 37 CFR 1.809(c)(d)and are monoclonal antibodies 2C5 (produced by a hybridoma deposited asATCC Accession No. PTA-11447), 2H9 (produced by a hybridoma deposited asATCC Accession No. PTA-11446), 2F10 (produced by a hybridoma depositedas ATCC Accession No. PTA-11450), 2E11 (produced by a hybridomadeposited as ATCC Accession No. PTA-11449) and 1F11 (produced by ahybridoma deposited as ATCC Accession No. PTA-11448), the production ofwhich is described in the Exemplification section, infra. The antibodiesdesignated 2C5, 2H9 and 2F10 bind specifically to different epitopes ofthe N-terminus of the osteopontin protein. The antibodies designated2E11 and 1F11 bind specifically to different epitopes of the C-terminusof the osteopontin protein.

The present invention is not limited to any particular assay format.Many different assay formats exist which may be useful in the presentinvention. For example, the assay format may be that of an Enzyme LinkedImmunoSorbent Assay (ELISA). ELISAs are designed for detecting andquantitating substances such as peptides, proteins, antibodies andhormones. Other names, such as Enzyme ImmunoAssay (EIA), are also usedto describe the same or similar process. In an ELISA, an antigen must beimmobilized to a solid surface. The solid surface may be, for example,the surface of a Petri dish or a micro-carrier bead. The antigen is thencomplexed with an antibody that is linked to an enzyme. Detection isaccomplished by incubating this enzyme-complex with a substrate thatproduces a detectable product. The most crucial element of the detectionstrategy is a highly specific antibody-antigen interaction.

Most commonly, ELISAs are performed in 96-well (or 384-well) polystyreneplates, which will passively bind antibodies and proteins. It is thisbinding and immobilization of reagents that makes ELISAs so easy todesign and perform, as first described by Eva Engvall, et al.(Enzyme-linked immunosorbent assay. II. Quantitative assay of proteinantigen, immunoglobulin G, by means of enzyme-labelled antigen andantibody-coated tubes. Biochim Biophys Acta, 251(3):427-434, 1971).Having the reactants of the ELISA immobilized to the microplate surfacemakes it easy to separate bound from unbound material during the assay.This ability to wash away nonspecifically bound materials makes theELISA a powerful tool for measuring specific analytes within a crudepreparation.

A detection enzyme may be linked directly to the primary antibody orintroduced through a secondary antibody that recognizes the primaryantibody. It may also be linked to a protein such as, for example,streptavidin if the primary antibody is biotin labeled. The mostcommonly used enzymes are horseradish peroxidase (HRP) and alkalinephosphatase (AP). Other enzymes have been used as well. These include,for example, β-galactosidase, acetylcholinesterase and catalase. A largeselection of substrates is available for performing the ELISA with anHRP or AP conjugate. For HRP, Promega's (Madison, Wis.) TMB substrateand Sigma's (St. Louis, Mo.) CPS Chemiluminescent Peroxidase Substrate,are just two examples known in the art. Chromatographic andchemiluminescent substrates, among others, are also available for AP andare well known in the art. The present invention is not limited by thechoice of detection enzyme or substrate as long as they are compatiblewith each other. The choice of substrate depends upon the necessarysensitivity level of the detection and the instrumentation available fordetection (spectrophotometer, fluorometer or luminometer). ELISA assaysare well known in the art. In addition to the above formats they may bepreformed in microtiter plates, in microarrays, in assay tubes (e.g.,eppendorf tubes) or on micro-carrier suspensions, etc. ELISA assays maybe read manually, with plate readers or the assay may be automated bythe use of, for example, robotics. Other assays are also useful in thepresent invention. For example, radioimmunoassays (RIA) may be used.These assays are also well known in the art (see, e.g., Boden and Chey,“Preparation and specificity of antiserum to synthetic secretin and itsuse in a radioimmunoassay (RIA),” Endocrinology, 92(6):1617-1624, 1973).

Still other assay formats exist that may be used with the presentinvention. For example, immuno-affinity chromatography may be used. Inone example, the capture antibody is bound to the column matrix. Asample is passed through the column and the column washed. A detectingantibody is then passed over the column and washed. The determiningantibody is then passed over the column and washed. The presence ofosteopontin fragments can then be determined based on the amount ofdetecting and determining antibodies recovered or by labeling asdiscussed both supra and infra. Those skilled in the art will know ofother suitable assay systems for use with the present invention.

The antibodies bound to the suspected osteopontin fragment may bedetected by any means known in the art. The present invention is notlimited to any particular detection means for detecting bound or unboundantibodies. In addition to the means discussed above, biotin-avidinsystems are known in the art and can be used with the present invention.Chromatographic, chemiluminescent and fluorescent detection systems arealso known in the art and can be used with the present invention.Detection systems may be either direct (with the detection moleculeconjugated directly to the primary antibody, i.e., the capture detectionor determining antibody of the present invention) or indirect (with thedetection molecule conjugated to a secondary antibody that hasspecificity for the primary antibody).

The reagents and methods of the present invention are useful in thedetection of various diseases and determining the progression andprognosis of those diseases. The present invention is not limited to anyparticular disease so long as osteopontin fragments are known orsuspected of being an indicator of disease presence, progression and/orprognosis. Some diseases in which the generation of osteopontinfragments is known or suspected of being an indicator of diseasepresence, progression and/or prognosis are breast cancer, prostatecancer, and lung cancer. Determination of the progress of a disease maybe accomplished, for example, by testing for the presence of osteopontinfragments over time with the methods of the present invention. Based onthe how the disease has progressed, as determined by the methods of thepresent invention, one skilled In the art will be able to generate aprognosis of the disease.

More specifically, the present invention also relates to reagents andmethods for the detection of cancer in a subject as well as formonitoring the progression and prognosis of the disease. In oneembodiment, the present invention detects cancer by measuring the levelof osteopontin fragments in a bodily fluid derived from the subjectusing the reagents and methods of the present invention. The level ofosteopontin may be determined over time to monitor the progression ofthe disease (either as it increases in severity or as it responds totreatment(s)) and determine the prognosis of the disease based onchanges in the level of osteopontin fragments and/or a change in thefragment sizes in the subject sample over time and/or as compared tohistoric data. The Exemplification section of the present application,below, provides details of non-limiting examples of how the reagents andmethods of the present invention may be used for detection, monitoringthe progression and determining the prognosis of subjects that have orare suspected of having diseases associated with the generation ofosteopontin fragments.

EXEMPLIFICATION Materials and Methods

Production of Monoclonal Antibodies. The following proteins wereprovided by Dr. Lucy Liaw (Maine Medical Center Research Institute,Portland, Me.): h-FL OPN (E. coli expression), GSTOPN(OPN with GST tagcleaved) (E. coli expression), m-FL OPN (E. coli expression), N-terminalOPN (E. coli expression) and C-terminal OPN (E. coli expression).Control His-tagged swine viral protein (+21×His-tag) was generouslyprovided by Idexx (Portland, Me.).

All centrifugations were performed at 1,000 rpm for 10 minutes unlessnoted otherwise. All media and buffers used were at room temperatureunless noted otherwise. The entire experimental procedure was carriedout at room temperature. Incubation was performed in a 37° C. incubatorfilled with 5% CO₂.

Immunization. Pre-immunization blood was collected the retro-orbitallyfrom an 11-month old female C57 BL OPN-null mouse (1284: OPN−/−)provided by Dr. Lucy Liaw. Immunizations were started at Day 0 by asubcutaneous injection of 100 μg of h-FL OPN suspended in 500 μl of CFA(Complete Freud's Adjuvant; Sigma, St. Louis, Mo.: Cat. No. F5881),followed by subsequent injections each with 50 μg of h-FL OPN in 500 μlof IFA (Incomplete Freud's Adjuvant; Sigma Cat. No. F5506) on Day 13,Day 33, Day 45 and Day 161. At Day 170 the mouse was bled and theantibody titer was measured by ELISA (see, below). Twenty-two days afterthe bleeding test a final injection was administered with 30 μg h-FL OPNin 150 μl IFA in the peritoneal cavity and with 20 μg in 100 μl IFAsubcutaneously. Four days later the mouse was bled, sacrificed and thespleen collected (Table 1).

TABLE 1 Mouse Identification and Immunization Steps Mouse Strain MouseID# DOB Sex Parents C57 BL 1284 Apr. 05, 2004 Female 994 X 1078 OPN-nullImmunization Day Route Comments: 0 SC Pre-immunization bleed, 100 μgpurified recombinant FL human OPN with Complete Freund's Adjuvant 13 SC50 μg purified recombinant FL human OPN with Incomplete Freund'sAdjuvant 33 SC 50 μg purified recombinant FL human OPN with IncompleteFreund's Adjuvant 45 SC 50 μg purified recombinant FL human OPN withIncomplete Freund's Adjuvant 161 SC 50 μg purified recombinant FL humanOPN with Incomplete Freund's Adjuvant 170 Bleed (retro-orbital) 192 IP &SC 50 μg purified recombinant FL human OPN with Incomplete Freund'sAdjuvant 197 Bleed (retro-orbital), spleen collected, cell fusionstarted

Antibody Titration Using ELISA. After five rounds of immunization 0.1 mlblood was collected from mouse eyes in order to determine the OPNantibody production level in the serum. A 96-well microtiter plate (BDBiosciences, Bedford, Mass.: Cat. No. BD353279) was coated with 50 ng/ml(100 μl/well) of purified h-FL OPN and incubated at 4° C. overnight.Subsequently, the plate was blocked with 3% nonfat dry milk in PBS-T(Phosphate Buffer Saline-Tween; PBS pH 7.3, 0.05% Tween 20) at roomtemperature for 2 hours. The sample serum was then loaded to the firstcolumn of the plate at 1 ul to 99 μl of 3% nonfat dry milk in PBS-T,followed by serial 5-fold dilutions. After incubation at 37° C. for 1hour the plate was washed 3× with PBS-T and incubated at 37° C. for 2hours with a combination of two separate secondary antibodies at 100μl/well. One of the secondary antibodies was a 1:5000 diluted Peroxidase(HRP)-conjugated Goat Anti-Mouse IgG, Fc_(γ) fragment specific (JacksonImmunoResearch Laboratories, West Grove, Pa.: Cat. No. 66350) and theother was a 1:5000 diluted Peroxidase (HRP)-conjugated AffiniPure GoatAnti-Mouse IgG+IgM (H+L) (Jackson ImmunoResearch Laboratories; Cat. No.66877). Use of the two secondary antibodies allowed the detection ofboth IgG and IgM types of antibodies against OPN.

Finally, the plate was washed 4 times with PBS-T, followed by incubationwith 100 μl/well of TMB (Tetramethyl benzidine) at room temperature indarkness for 15 minutes. The chromogenic reaction was stopped by 100μl/well of 1 M HCl. The amount of the antibodies against h-FL OPN wasdetermined by reading the plate at 405 nm using an automated microplatereader (Bio-Tek Instruments).

Fusion of Splenocytes and Myeloma Cells. The hybridoma cells producingantibodies against OPN were generated by fusing splenocytes with myelomacells according to a standard protocol developed by Kohler and Milstein(Kohler G, Milstein, C, 1975). The FO myeloma cells were a gift fromHatim Chraibi (Maine Biotechnology Services, Portland, Me.; see, alsoATCC, Bethesda, Md.: Cat. No. CRL-1646; de StGroth and Scheidegger, JImmunol Methods, 35(1-2):1-21, 1980). These cells were harvested andwashed in mid-log phase with a cell concentration of 9.5×10⁵ cells/ml atthe time of fusion.

The spleen from the immunized mouse was first removed and washed 3 timeswith 2 ml of serum-free DMEM/F12 (Mediatech, Inc, Herndon, Va.: Cat. No.10-090-CV). It was then placed in a Petri dish with a few drops ofserum-free DMEM/F12. After smashing the spleen between two fine sterilescreens the splenocytes were separated and collected by rinsing thePetri dish and the screens 3 times with 5 ml of serum-free DMEM/F12.These cells were then centrifuged and resuspended in 4.5 ml of cold redblood cell lysing buffer (17 mM tris-HCl, pH 7.2, 0.144 M NH₄Cl), andsit at room temperature for 5 minutes. After addition of 10 ml of coldDMEM/F12 they were centrifuged again and resuspended in 20 ml serum-freeDMEM/F12. A total of 8.2×10⁷ cells collected.

The cell fusion was achieved by combining these two cell populations ata 5:1 (splenocyte:myeloma) ratio. Following washing and centrifugation,2 ml of pre-warmed 50% PEG (Sigma, St. Louis, Mo.: Cat. No. 72K2300) wasadded to the cell pellet in a drop by drop fashion over 1 minute periodwhile swirling the tubes gently and the suspension was stirred with a1-ml glass pipette for an additional minute. Subsequently, 5 ml ofserum-free DMEM/F12 were added in the same fashion over a 2 minuteperiod immediately followed by the addition of another 5 ml ofserum-free DMEM/F12. The cell suspension was then allowed to sit at roomtemperature for 3 minutes before receiving 40 ml of HT (hypoxanthine andthymidine) medium (DMEM/F12, 10% FBS, Omega Scientific: Cat. No. FB-02;1% penicillin/streptomycin, Cambrex: Cat. No. 17-602E; 1% L-Glutamine,Cambrex: Cat. No. 17-605E; 1×HT supplement, Gibco: Cat. No. 11067-030;10% NCTC, Cambrex: Cat. No. 12-923E; 0.01% Insulin-Transferrin-Selenium,Gibco: Cat. No. 41400-045) to achieve a total volume of 50 ml. The cellswere centrifuged at 700 rpm for 7 minutes, washed and resuspended in 40ml HT medium to achieve a final concentration of 7×10⁵ myeloma cells/ml.The fused cells were then incubated with culture flasks in an uprightposition at 37° C. and 5% CO₂ for 1.5 hours with 1.6 ml 50× Aminopterinadded at the end of incubation. Finally, the cell suspension wastransferred to 96-well tissue culture plates (BD Biosciences: Cat. No.353072) at 100 μl/well and incubated at 37° C. for 10 days.

Screening of Hybridoma culture Supernatants by ELISA. The hybridomaculture supernatants were initially screened with 100 ng/ml h-FL OPNusing Peroxidase (HRP)-conjugated goat anti-mouse IgG+IgM (H+L) as thesecondary antibody. In the follow-up screenings GSTOPN (OPN with GST tagcleaved), a recombinant human OPN without His-tag, was used in additionto h-FL OPN as the positive antigens. These screenings also includedcontrol His tagged swine viral protein and 1×PBS as negative controlsalong with Peroxidase (HRP)-conjugated goat anti-mouse IgG, Fc_(γ)specific as the secondary antibody.

The ELISA was conducted essentially as described above. The 96-wellmicrotiter plate was coated with 50-100 ng/ml of antigen and a 1:5000diluted HRP-conjugated goat anti-mouse antibody was used as thesecondary antibody.

If a well was tested positive with both h-FL OPN and GST as antigen andwith HRP-conjugated Goat anti-mouse IgG,Fc_(γ) as secondary antibody thecorresponding hybridoma culture was expanded and retested for threetimes. Three primary hybridoma cultures—1284:4E8, 1284:2B11, and1284:3G11—were selected for subcloning by limiting dilution 18 daysafter fusion. The rest of the positive primary hybridoma cell lines werefrozen and stored in liquid Nitrogen.

Rescue the Low Antibody Activity Hybridomas. ELISA testing showed thatHybridomas 1284:4F8 and 1284:1H4 had low antibody activities. In anattempt to boost their antibody yield a rescue method based on seriallimiting dilution was employed to isolate and enrich the hybridoma cellssecreting anti-OPN antibodies. Initially, the cells were grown to logphase, counted, diluted with HT medium to 5000 cells/100 μl and grown ina 96-well plate at 37° C. for 2-3 days. Subsequently, the hybridomasupernatants were screened with ELISA and the cells that produced strongsignals (rated “++” or higher as in Table 2) were collected andsubjected to the above steps at serially increasing densities, i.e., at500, 100 and 10 cells/100 μl. In Table 2 a list of polyclonal primaryhybridomas secreting anti-OPN antibodies developed from OPN immunizedmice 1284 and 1283. The number of “+” symbols is an indicator ofantibody activity level. “Y” indicates the location of anantibody-binding site (i.e., an epitope) on the OPN fragment. Theantibody activities for 1284:4F8 and 1284:1H4 were the results afterrescue. Finally, the cell cultures were expanded and stored in liquidnitrogen.

TABLE 2 ELISA Screening of Primary Hybridomas Positive for anti-OPNantibodies. Antibody activities N - Terminal C - Terminal Hybridoma(against h-FL OPN) OPN specificity OPN specificity 1283: 1C12 +++ 1283:2G8 +++ Y 1284: 4E8 +++ Y 1284: 2B11 +++ Y 1284: 3G11 +++ Y 1284: 2C10+++ Y 1284: 4F8 +++ Y 1284: 1H4 ++ +++: A_(405 nm) = 0.8-2.0, ++:A_(405 nm) = 0.5-0.8, +: A_(405 nm) < 0.5

Subcloning. The two hybridoma cell lines, 1283:1C12 and 1283:2G8, weregenerated previously in our laboratory. These two cell lines producedmouse polyclonal antibodies against h-FL OPN. After being thawed at 37°C. they were tested for their antibody-producing activities as describedabove.

A total of five cell lines, 1283:1C12, 1283:2G8, 1284:4E8, 1284:2B11 and1284:3G11, were grown to log phase before being harvested, counted anddiluted with HT medium containing 10% hybridoma cloning factor (BioVerisCorp, Gaithersburg, Md.: Cat. No: 210001) to 0.5-1 cell/200 μl. Thediluted cells were then plated at 0.5-1 cell/well and observed forcolony growth from a single clone after a 3-day incubation period. At10-14 days into incubation positive subclones were identified by ELISAand then expanded and frozen in liquid nitrogen. The corresponding cellsupernatants were stored at −20° C. Three of the five cell lines,1283:1C12, 1283:2G8, and 1284:4E8, were subjected to this subcloningprocedure for 2-3 times until a true monoclone from each cell line wasidentified.

Production of Ascites Fluid. The strain of mouse used in the ascitesproduction was 6- to 8-week old OPN heterozygotes with H-2^(b/d) MHCmolecules, which were offspring from a cross between the C57/BL H-2^(b)and the Balb/c H-2^(d). These heterozyzotes were used to produce acsitesfluid because the anti-OPN hybridoma cells were generated by fusing theOPN^(−/−) C57/BL mouse splenocytes with Balb/C mouse FO cells. Thesehybridoma cells, carrying MMC H-2^(b/d) molecules, could only grow inmice with the same MHC background.

F1 hybrid offsprings of the C57/BL and Balb/c mice were first primedwith an intraperitoneal injection of 500 μl of IFA. Seven to ten dayslater 5×10⁵-5×10⁶ washed hybridoma cells were injected into theperitoneum. When ascites was evident the mice were sacrificed forascites fluid collection. Ascites production was approved and supervisedby the Maine Medical Research Institutional Animal Care Use Committee.

Purification of Ascite Fluid. After the ascites fluid was collected(see, above), the lipids and cell debris were removed by centrifugationat 14000 g for 15 minutes. The purified ascites fluid sample was dilutedby adding 5 volumes of the binding buffer (20 mM sodium phosphatebuffer, pH 7.0).

Preparation of Protein G Column. The Protein G Resin Slurry (ImmobilizedProtein G, Pierce Cat. No. 20398) was poured into a gravity-flow column(a gift from the Foundation of Blood Research, Portland, Me.) to a gelbed volume of 1 ml for each ascite sample. The columns were equilibratedby adding 5 ml of the binding buffer and the solution was allowed todrain through the column.

Purification of the MAbs. After the diluted samples were loaded andcycled through the column for three times. The column was washed with 5ml of binding buffer to remove any unbound proteins and the wash wascollected in 1-ml aliquots (labeled as protein G Flow Thru). Theantibodies were eluted with 5 ml of elute buffer (0.1 M Glycine-HCL, pH2.7), collected in 1-ml fractions and immediately adjusted tophysiologic pH by adding neutralization buffer (1 M Tris-HCL, pH 8.0).The protein G column was re-equilibrated with 5 ml binding buffer.

The quantities of the proteins were determined using a DC protein assaykit (Bio-Rad Laboratories, Hercules, Calif.: Cat. No. 500-0116). Onaverage, 1-4 mg of antibodies were produced from 1 ml of ascite fluid.Purities were assessed by SDS-PAGE and coomassie blue staining Briefly,10 μg/lane of reduced antibody samples were resolved on a 12.5%polyacrylamide gel following the procedure given above. The gel was thenstained in Coomassie blue (0.025% Coomassie brilliant blue 8250, 40%(v/v) methanol, 7% (v/v) acetic acid) at room temperature overnight,followed by destaining with destain solution I (40% (v/v) methanol, 7%(v/v) acetic acid) for 1 hour and with destain solution II (5% (v/v)methanol, 7% (v/v) acetic acid).

Dialysis. The eluted antibody fractions were loaded into a 12,000-14,000MWCO (molecular weight cut-off) Spectra/Por molecularporous membranedialysis tubing (Spectrum, VWR) and dialyzed in >100 volumes of PBSbuffer (pH 7.3) at 4° C. for 36 hours. The dialysis buffer was changed 3times during dialysis.

Biotin-NHS Biotinylation of Antibodies (1F11, 2F10 and 1E3).Sulfo-NHS-LC-Biotin [sulfosuccinimidyl-6-(biotinamido) hexanoate] (MW556.59, Pierce) was prepared at 10 mg/ml in PBS and added to dialyzedMAbs 1F11, 2F10 and 1E3 at a ratio of 186 μg, 148 μg and 150 μg ofbiotin per milligram of antibody, respectively. 1E3, a monoclonalantibody against platelets activation antigen CD62-P (P-selectin) wasused as a control.

The mixtures were incubated at room temperature for 1 hour before beingdialyzed extensively against PBS to remove uncoupled biotin. Thequantities of the biotinylated proteins were then determined using a DCprotein assay kit (Bio-Rad Laboratories, Hercules, Calif.).

Characterization of Monoclonal Antibodies (MAbs)

Isotyping of MAbs 2E11, 2C5, 2H9, 1F11 and 2F10. The isotypes of 2E11,2C5, 2H9, 1F11 and 2F10 were determined with the IsoStrip MouseMonoclonal Antibody Isotyping Kit (Roche, Indianapolis, Ind.: Cat. No.1493027), following manufacturer's instructions. Briefly, 150 μl of aneat (unpurified) antibody supernatant sample was added to a developmenttube (included in the kit) and briefly agitated to resuspend the coloredlatex particles completely. An isotyping strip was then inserted intothe development tube with the black end at the bottom and incubated for5-10 minutes. The appearance of a blue band in the class or subclasssection of the strip, as well as in the κ or λ section, was anindication of the antibody's class or subclass and light-chaincomposition.

Indirect ELISA. A 96-well flat bottom ELISA plate was coated with 100 μl(200 ng/ml in PBS pH 8.0) of antigens at 4° C. overnight. Depending onthe purpose of the experiment, different antigens were used:

For antibody specificity test, the wells were coated with h-FL OPN,GSTOPN, control His-tagged swine viral protein and 1×PBS (pH 7.3),respectively, with the latter two as negative controls.

For antibody species cross-reaction test, the wells were coated withh-FL OPN and m-FL OPN, respectively.

For antibody epitope mapping test, the wells were coated with h-FL OPN(62 KDa) and its N- and C-terminal fragments (40 KDa for N-terminal and25 KDa for C-terminal), respectively.

The rest of the ELISA procedure was carried out as described aboveexcept after blocking supernatants of the hybridoma culture fluids orpurified ascite fluids were added to each well with or without dilutionfollowed by 2-hour incubation at 37° C.

SDS-PAGE and Western Blot Analysis. The sodium dodecylsulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) was performed witha Mini-PROTEAN Gel Electrophoresis Unit (Bio-Rad Laboratories, Hercules,Calif.). Protein samples were diluted appropriately and boiled for 5minutes in the sample buffer (63 nM Tris, 2% SDS, 0.01% bromphenol blue,5% β-mercaptoethanol). For samples of h-FL OPN, m-FL OPN, N-terminal OPNand C-terminal OPN, 20 ng of protein were loaded in each lane of a 12.5%polyacrylamide gel. For tissue lysates, 10-20 μg/lane of proteins wereused. Proteins were separated with 100 V constant voltage using thebuffer system of Laemmli (Laemmli, U. K. 1970. Cleavage of structuralproteins during the assembly of the head of bacteriophage T4. Nature227:680-685; Biorad Instruction Manual) for approximately 1-2 hours. Therunning buffer consisted of 25 nM Tris base, 192 mM glycine, 0.1% SDS,pH 8.8.

The separated proteins on the gel were transferred to a polyvinylidenedifluoride (PVDF) membrane with the Mini-PROTEAN Gel ElectrophoresisUnit (Bio-Rad Laboratories) running at 350 mA for 1-2 hours. Thetransfer buffer consisted of 25 mM Tris, 192 mM Glycine and 0.1% SDS, pH8.3 (Biorad Instruction Manual).

Nonspecific binding was blocked with 5% nonfat dry milk in TBS-T buffer(10 nM Tris Base, pH 8.0, 150 nM NaCl, and 0.05% Tween 20) at roomtemperature for 1 hour. The membrane was then incubated in a neat MAbhybridoma culture supernatant or a diluted purified MAb (1:500) in 5%milk TBS-T. After overnight incubation at 4° C., the membrane was washed5 times with TBS-T. The secondary antibody, HRP-conjugated goatanti-mouse IgG, Fc₇, (1:10,000) in 5% milk TBS-T (Tris-Buffer Saline,Tween), was then added to the membrane. After 1-hour incubation and 5washings with TBS-T, the OPN bands were visualized using a chromogenicTMB substrate (provided by Binax, Scarborough, Me.) and a CN/DABsubstrate Kit (Pierce, Rockford, Ill.: Cat. No. 34000) with a 10-minuteincubation in darkness. The reaction was stopped by rinsing the membranein distilled water.

Alternatively, an ECL Western blotting detection reagent (Amersham,Chicago, Ill.: Cat. No. RPN2108) was used and the Western blot resultsdeveloped on a Kodak X-ray film. Prestained molecular weight standards(Bio-Rad, Hercules, Calif.: Cat. No. 161-0305) were used to estimateprotein sizes.

Indirect Competition ELISA. To compare epitope specificity, unlabeledanti-OPN MAbs were used to compete with biotinylated 1F11 and 2F10 forbinding to specific OPN epitopes. Briefly, a 96-well plate was coatedwith 200 ng/ml (100 μl/well) h-FL OPN and blocked with 3% non-fat drymilk with PBST for 1 hour at 37° C. Subsequently, 100 μl/well ofPBS+milk (blank, negative control), UCD/PR (negative control) andmonoclonal antibodies 2E11, 2C5, 2H9, 1F11 and 2F10 culture supernatantswere added, followed by incubation at 37° C. for 1-2 hours. After theplate was washed with 3×PBS-T, 100 μl/well 1:500 diluted biotinylated1F11 (0.66 μg/ml) or 1:500 diluted biotinylated 2F10 (1.43 μg/ml) wereadded and incubated at 37° C. for 1 hour. Following wash with 3×PBS-T,the plate was incubated with 100 μl/well 1:1000 diluted HRP-conjugatedStreptavidin (Jackson ImmunoResearch Laboratories, West Grove, Pa. Cat.No. 55889) for 1 hour at 37° C. The rest of the ELISA steps wereperformed as described above

Tissue Extracts. Kidney and skull tissue extractions from rats and micewere used to assess the reactivity of the MAbs with native OPN. The rattissue extracts were provided by Dr. Volkhard Lindner (Maine MedicalResearch Institute, Portland, Me.). Extracts from wild-type,OPN-deficient mice and OPN heterozygotes (described above) were used aspositive and negative controls for Western blots, respectively. Briefly,after the mice were sacrificed, the skulls and kidneys were removed andimmediately frozen in liquid nitrogen. The frozen tissue was manuallyground into powder and suspended in lysis buffer (0.05 M Tris pH 7.6, 1%SDS, 0.001 M PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin) andsonicated. The suspension was centrifuged at 15,000 g for 5 minutes andthe supernatant was retained. The quantities of the extract proteinswere determined using a DC protein assay kit. Western blots wereperformed as described above.

Immunohistochemistry. A 15-day old wild-type mouse embryo was fixed inperiodate-lysine-paraformaldehyde (PLP) at 4° C. for 24-48 hours,cryoprotected in 20% sucrose in PBS at 4° C. overnight and then frozenin OCT compound (a low-temperature embedding medium for cryosectioningtechniques, Leica, Bannockburn, Ill.). The tissue sections (5 μm inthickness) were prepared with a Cryocut 1800 tissue sectioner (Leica),and stored at −70° C. until use.

Before immuno-detection, the slides were dried at 60° C. for 20 minutesand rinsed in tap water to wash off the tissue embedded compound OCT,followed by baking at 60° C. for another 20-30 minutes and rinsing inPBS. Antigens were retrieved from the cells by steam boiling the tissuesections in citrate buffer (pH 6.0, 0.01 M citric acid monohydrate, 0.01M Sodium Citrate Tribasic dehydrate, 0.05% Tween 20) for 45 minutes. Theendogenous peroxidase activity was eliminated by incubation with 0.3%hydrogen peroxide in a moist chamber for 30 minutes. Nonspecific bindingwas blocked by incubation with 2% normal goat serum in PBS containing 1%BSA (PBS-BSA), which was also used to dilute all subsequent antibodies.

At immuno-detection stage, antibodies were incubated with specimens for2 hours at room temperature at the following final concentrations:purified mouse IgG (negative control), 1 μg/ml; biotinylated 1F11, 3.3μg/ml; biotinylated 2F10, 7.18 μg/ml. The negative control slide waswashed 5 times for 5 min each in PBS-T and then incubated with 1:1000HRP-conjugated goat anti-mouse antibody at room temperature for 1 hour.The test sample slides were washed 5 times for 5 min each in PBS-Tbefore signal detection with the ABC Elite kit (Vector Laboratories,Inc.: Cat. No PK-6105) and diaminobenzidine (DAB) as substrate.Conventionally stained slides (with methylene blue) were examined underlight microscopy.

Cell Adhesion Assay. The optimal dosage of OPN as a cell-adhesionpromoting factor was determined by following a procedure from Liaw, etal., 1994. In brief, a 96-well Maxisorp plate (Nunc, Naperville, Ill.)was coated with 100 μl/well of h-FL OPN that was 2-fold serially dilutedfrom 500 nmol to 3.9 nmol. 1×PBS coated wells were used as negativecontrol (0.0 nmol). The mouse embryonic fibroblasts cell line, MEF; themouse mammary tumor cell line, PyVT and the non-invasive breast cancercell line, MCF-7 (kindly provided by Dr. Lucy Liaw, MMCRI, Maine) wereadded to the plate at approximately 30,000 cells/well. After adhesion at37° C., 5% CO₂ for 30-90 minutes, non-adhering cells were rinsed offwith PBS and the adhering cells were fixed with 4% paraformaldehyde,stained with 0.5% toluidine blue, solubilized with 1-10% SDS and read ina microtiter plate reader at 595 nm.

After the optimal OPN dosage was determined, the inhibitive effect ofthe anti-OPN antibodies to cell adhesion was tested with two cell lines,MEF and PyVT. Briefly, a 96-well plate was coated with h-FL OPN at 62.5nM and the anti-OPN antibodies were added to the wells 30 minutes beforecell adhesion. A monoclonal antibody against CD62-P (IE3) was used asthe negative control.

Papillary Thyroid Carcinoma Patient Plasma. Fourteen PTC plasma sampleswere collected from patients who have been diagnosed with papillarythyroid carcinomas (PTC) (effort of Dr. Al Driedger, London HealthScience Center, London, Ontario, Canada). Group 1 consists of foursamples from patients with less than 1 ng/ml blood thyroid globulin(Tg). Group 2 consists of the other 10 samples from patients waiting for¹³¹I radiation therapy. The blood was collected in EDTA(ethylenediaminetetraacetic acid); the plasma were separated and frozenat −80° C. within 4 hours of collection.

Kidney Transplant Patient Plasma and Urine Samples. Sixty-one kidneytransplant patient samples, including 26 urine samples (3 pre-transplantand 22 post-transplant samples) and 35 plasma samples (13 pre-transplantand 22 post-transplant samples) were generously provided by Dr. JohnVella, Nephrology and Transplantation Center, Maine Medical Center,Portland, Me. All patients had kidney diseases and were qualified forthe kidney transplant. Plasma was also collected from 21 healthyvolunteer blood donors who formed the control group.

Sandwich (Two-Antibody) ELISA for OPN Measurement. A two-antibodySandwich ELISA was developed as a quantitative ELISA for OPNmeasurement. MAb 2F10, which binds specifically to the N-terminal ofOPN, was selected as the capture antibody; while 1F11, specific for theC-terminal, was selected as the determination antibody (1F11 wasbiotinylated). This assay design aimed to identify only relativelyintact OPN molecules that were in a sample.

Initially, chessboard titration experiments (Crouther, et al., volume149; setting up the assay in a “chessboard” pattern takes into accountthe variability inherent to the assay plate and the detection system)were carried out to determine the optimal concentrations of captureantibody 2F10 and detection antibody 1F11. It was determined that 2F10at 1.5 μg/ml and 1F11 at 0.6 μg/ml would be the optimal antibodyconcentrations for the sandwich ELISA.

This assay was then performed by first establishing an OPN-dosageresponse curve (standard curve), which was used to determine the OPNquantity in a sample. All samples were run in triplicates and undiluted.The capture antibody, 2F10, was diluted 1:500 (1.5 μg/ml) in PBS andadded to a 96-well flat bottom ELISA plates (BD Falcon, VWR) at 100μl/well. After coating at 4° C. overnight, the plate was rinsed twicewith PBS-T before being blocked with 3% nonfat milk in PBS-T at 37° C.for 1 hour.

Subsequently, 100 μl of serially diluted OPN standards or 50 μl ofpatient plasma or urine samples were added to each well followed by1-hour incubation at 37° C. The standards were comprised of 100 μl/wellof h-FL OPN 2-fold serially diluted from 5.0 to 0.002 μg/ml. After 4×washing with PBS-T, the detection antibody, biotinylated 1F11 diluted1:500 (0.6 μg/ml) in 3% nonfat milk in PBS-T, was added and the platewas incubated at 37° C. for 2 hours. Following the second washing, 1:500(2 μg/ml) diluted HRP-conjugated streptavidin was added and the plateincubated at 37° C. for 1 hour. After the final wash, TMB substrate wasadded to develop color.

Results

Antibody Titration for Serum from Immunized Mouse. The serum obtainedfrom mouse strain 1284 on Day 170 after five immunizations was subjectedto ELISA testing to determine the activity of the anti-OPN antibodies.The titer was 3.125×10³, indicating that the mouse had producedsufficient anti-OPN antibodies and it was ready for using in fusionexperiment (FIG. 1).

Hybridoma Screening. In order to identify those hybridoma culturesproducing anti-OPN antibodies, an initial ELISA screening of thehybridoma supernatants was performed after 14 days of fusion. Sixhybridoma cultures were identified secreting antibodies (IgG or IgM)against human full-length (h-FL) OPN, and four of them (1284:4E8,1284:2B11, 1284:3G11 and 1284:2C10) produced high antibody activities.The other two hybridomas (1284:4F8 and 1284:1H4) initially producedantibodies at lower activities; nevertheless, these two hybridomas wereable to achieve satisfactory antibody levels after further screeningprocedures (see, Materials and Methods): from “+” to “+++” for 1284:4F8and from “+” to “++” for 1284:1H4 (Table 2).

In order to verify which types of antibodies they were producing, thesesix hybridoma cultures were expanded and the second ELISA screening wasperformed with three control antigens including GSTOPN (cleaved humanfull-length OPN), control His-tagged swine viral protein, and 1×PBS,along with HRP-conjugated goat anti-mouse IgG (Fc_(γ) specific) as thesecondary antibody. The results showed that all six primary hybridomassecreted IgG against OPN.

Subcloning. Five randomly selected hybridoma cultures (1283:1C12,1283:2G8, 1284:4E8, 1284:2B11 and 1284:3G11) were subcloned immediatelyby limiting dilution to generate monoclones. Two of them, 1283:1C12 and1283:2G8, were produced previously in our lab. These two hybridomas wereprepared from frozen stocks. After 1-3 times of repetitive subcloning,25 monoclones were obtained from the five primary hybridoma cultures(Table 3), five of which (2F9, 2C5, 2H9, 1F11 and 2F10) were appearedpromising and subjected to further characterization.

Development hierarchy of the anti-OPN monoclonal antibodies (Table 3).The single cell subcloning technique was applied to 1283:1C12, 1283:2G8,1284:4E8, 1284:2B11, and 1284:3G11 primary hybridoma cultures, obtainedafter the fusion experiments, which showed strong antibody activities inELISA. Circled names indicate monoclones. Bold italic fonts indicate thefive monoclones selected for further studies.

For example, in the first subcloning procedure for cell culture1283:1C12, cells from well 2F5 were subjected for the second subcloning.Subsequently, cells from well 1D1 were chosen for the third round,resulting in monoclones 1C9, 2B3, 2E11, and 2F9. Finally, 2F9 wasselected for further studies and the other three clones were cryopreserved in liquid nitrogen.

TABLE 3 Development Hierarchy of the Hybridomas Producing Anti-OPNMonoclonal Antibodies

Production of Ascite Fluid. Because the FO myeloma cells (e.g., ATCC,Bethesda, Md.: Cat. No. CRL 1646) were derived from a Balb/cJ mouse andsplenocytes were from OPN—null mice of C57BL origin (H-2^(b)), aheterozygous mouse strain (H-2^(b/d)) crossed from the C57BL and theBalb/cJ mice, as well as an unrelated athymic nude strain, were used forascite fluid production.

Information regarding each mouse used for ascite fluid collection, aswell as the total volumes of acsite fluid collected for each monoclonalantibody is listed in Table 4. The total volumes of ascite fluidcollected from each MAb were 2E11: 28.8 ml; 2C5: 26.7 ml; 2H9: 30.3 ml;1F11: 31.5 ml; and 2F10: 43.0 ml.

TABLE 4 Collection of Ascite Fluids Mouse Strain DOB Sex Parents MouseID MAbs Ascite Volume (ml) Sum (ml) Balbc/JxOPN−/− C57B46 Jan. 23, 2006female Balbc/Jx1940 2250 1F11 3 31.5 Balbc/JxOPN−/− C57B46 Jan. 23, 2006female Balbc/Jx1940 2251 1F11 4.5 Balbc/JxOPN−/− C57B46 Jan. 23, 2006male Balbc/Jx1940 2252 1F11 3 Balbc/JxOPN−/− C57B46 Jan. 23, 2006 maleBalbc/Jx1940 2253 1F11 1.5 Balbc/JxOPN−/− C57B46 Jan. 23, 2006 maleBalbc/Jx1940 2254 1F11 4.5 Balbc/JxOPN−/− C57B46 Mar. 13, 2006 femaleBalbc/Jx1948 2352 1F11 4.5 Balbc/JxOPN−/− C57B46 Mar. 13, 2006 femaleBalbc/Jx1948 2353 1F11 6 Hsd-Athymicnude-Foxninu Feb. 7, 2005 maleHarlan 34 1F11 4.5 Balbc/JxOPN−/− C57B46 Dec. 28, 2005 femaleBalbc/Jx1781 2235 2C5 7 26.7 Balbc/JxOPN−/− C57B46 Dec. 28, 2005 femaleBalbc/Jx1781 2236 2C5 3 Balbc/JxOPN−/− C57B46 Dec. 28, 2005 femaleBalbc/Jx1781 2237 2C5 2.5 Balbc/JxOPN−/− C57B46 Dec. 28, 2005 femaleBalbc/Jx1781 2238 2C5 2.7 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 maleBalbc/Jx1948 2241 2C5 1.5 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 maleBalbc/Jx1948 2242 2C5 2 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 maleBalbc/Jx1948 2243 2C5 1.5 Hsd-Athymicnude-Foxninu Feb. 7, 2005 maleHarlan 32 2C5 6.5 Balbc/JxOPN−/− C57B46 Dec. 28, 2005 male Balbc/Jx17812233 2E11 7.5 28.8 Balbc/JxOPN−/− C57B46 Dec. 28, 2005 male Balbc/Jx17812234 2E11 8 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 male Balbc/Jx1948 22402E11 euthanized Balbc/JxOPN−/− C57B46 Mar. 13, 2006 male Balbc/Jx19482347 2E11 2 Balbc/JxOPN−/− C57B46 Mar. 13, 2006 male Balbc/Jx1948 23482E11 4 Hsd-Athymicnude-Foxninu Feb. 7, 2005 male Harlan 31 2E11 2.8Hsd-Athymicnude-Foxninu Feb. 7, 2005 male Harlan 35 2E11 4.5Balbc/JxOPN−/− C57B46 Jan. 23, 2006 male Balbc/Jx1948 2255 2F10 5 43Balbc/JxOPN−/− C57B46 Jan. 23, 2006 male Balbc/Jx1948 2256 2F10 5.5Balbc/JxOPN−/− C57B46 Jan. 23, 2006 male Balbc/Jx1948 2257 2F10euthanized Balbc/JxOPN−/− C57B46 Mar. 13, 2006 female Balbc/Jx1948 23432F10 4 Balbc/JxOPN−/− C57B46 Mar. 13, 2006 female Balbc/Jx1948 2344 2F104 Balbc/JxOPN−/− C57B46 Mar. 13, 2006 female Balbc/Jx1948 2345 2F10 1.5Balbc/JxOPN−/− C57B46 Mar. 13, 2006 male Balbc/Jx1948 2346 2F10 5Balbc/JxOPN−/− C57B46 Mar. 13, 2006 female Balbc/Jx1948 2349 2F10 4Balbc/JxOPN−/− C57B46 Mar. 13, 2006 female Balbc/Jx1948 2350 2F10 4.5Balbc/JxOPN−/− C57B46 Mar. 13, 2006 female Balbc/Jx1948 2351 2F10 5.5Hsd-Athymicnude-Foxninu Feb. 7, 2005 male Harlan 36 2F10 4Balbc/JxOPN−/− C57B46 Dec. 28, 2005 female Balbc/Jx1781 2239 2H9euthanized 30.3 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 male Balbc/Jx19482244 2H9 2.5 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 male Balbc/Jx1948 22452H9 2.5 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 female Balbc/Jx1948 2246 2H94.5 Balbc/JxOPN−/− C57B46 Dec. 30, 2005 female Balbc/Jx1948 2247 2H9 4.5Balbc/JxOPN−/− C57B46 Dec. 30, 2005 male Balbc/Jx1948 2248 2H9 1.5Balbc/JxOPN−/− C57B46 Dec. 30, 2005 female Balbc/Jx1948 2249 2H9 5Hsd-Athymicnude-Foxninu Feb. 7, 2005 male Harlan 33 2H9 9.8 Total Ascitevolume (ml) 160.3

Verification of MAbs Purity. The total volumes of the purified andbiotinylated MAbs and their antibody concentrations were listed in Table5. Antibodies 2E11, 2C5 and 2H9 were purified from 1 ml of each antibodyascite fluid. Antibodies 1F11 and 2F10 were purified from 2 ml ascitefluid. The final concentration of each antibody was 0.23 mg/ml, 1.4mg/ml, 0.40 mg/ml, 0.9 mg/ml and 0.75 mg/ml, respectively, with thetotal volume of 3 ml, 2.5 ml, 3.5 ml, 6.5 ml and 4.6 ml in 1×PBS,respectively. Only the 1F11 and 2F10 antibodies were subject tobiotin-labeling from 1 ml of each purified antibody. The finalconcentration of these biotinylated antibodies was 0.33 mg/ml and 0.718mg/ml with the volume of 1 ml and 0.8 ml, respectively.

TABLE 5 Information of Purified MAbs Concentration Volume Purified MAbs(mg/ml) (ml) 2E11 0.23 3 2C5 1.4 2.5 2H9 0.40 3.5 1F11 0.9 5.5 2F10 0.753.6 Biotinylated-1F11 0.33 1 Biotinylate-2F10 0.718 0.8

In order to determine the purities of the five MAbs—2E11, 2C5, 2H9,1F11, and 2F10—the samples were resolved with SDS-PAGE and stained withCoomassie blue. In brief, the anti-OPN MAbs were purified from ascitesfluids using a Protein G column. The samples were loaded onto a 12.5%SDS—polyacrylamide gel under reducing condition. After Coomassie bluestaining the two bands representing the heavy chain (˜55 KDa) and thelight chain (˜25 KDa) of IgG are clearly visible. A purified human IgGsample served as a positive control. MAb 1F11 shows a 55 KDa and a 25KDa bands (FIG. 2A) and MAb 2F10 shows a 55 KDa band (FIG. 2B). Each ofthe MAbs 2E11, 2C5, and 2H9 shows a 55 KDa and a 25 KDa band. Themolecular weight protein standards (Bio-Rad) are shown as indicated. AllMAbs except 2F10 showed a 55 kDa and a 25 kDa band, representing theheavy chain (˜55 kDa) and the light chain (˜25 kDa) of IgG, respectively(FIGS. 2A, B). Antibody 2F10 only displayed a band at 55 kDa (FIG. 2A).

Normalized Titration of Hybridoma Culture Supernatants, Purified MAbs,and Biotinylated MAbs. Large amounts of MAb supernatant and ascite fluidwere collected. The supernatant was collected by growing hybridoma celllines in a 175-cm² tissue culture flask until the culture media turnedyellow. The ascite fluid was collected by injecting hybridoma cells intothe mouse and purified using protein G column. In order to determine theactivities of different antibodies, titration testing was performedusing ELISA with 10-fold serially diluted antibodies. The resultingtiters were normalized based on their concentrations so that comparableamounts of antibodies could be used for subsequent experiments. FIGS. 3Aand B showed that the ascite fluid had much higher antibody activitiesthan the supernant had. For example, the titer for 2E11 in 100× dilutedascite fluid was ˜59 absorbance units per mg/ml antibody while in 10×diluted supernatant, it was ˜12 absorbance units per mg/ml antibody,which translated into a ˜50× higher antibody activity in the ascitefluid. This observation was consistent with the known fact that ascitefluid in general produces higher antibody concentrations thansupernatant. However, the 2C5 activity was low in both the supernatantand the ascite fluid (the titer was ˜3 absorbance units per mg/mlantibody in 100× diluted ascite fluid). The reason leading to the low2C5 activity was not clear.

After being purified from the ascite fluid, the majority of theantibodies activities were lost, probably because of proteindenaturation during elution (FIGS. 3 A-C). For example, purified 2E11titer was ˜14 absorbance units per mg/ml antibody in 10× diluted buffercompared to ˜59 absorbance units per mg/ml antibody in 100× dilutedascite fluid. Column leaching was unlikely because little or no proteinswere detected in the column flow-through. 2C5 lost all of its activityafter purification. Similarly, 1F11 still retained most of its activitywhile 2F10 lost almost all the antibody activity after biotinylation(FIG. 3C). This observation was consistent with a previous report that aloss of antibody immunoreactivity of some of the leukocyte surfaceproteins occurred after using NHS-Biotin to label cells (Yates, et al.,1988).

Isotyping of MAbs 2E11, 2C5, 2H9, 1F11 and 2F10. The immunoglobulinisotype of each MAb was determined using IsoStrip Mouse MonoclonalAntibody Isotyping strip. The isotype of the MAbs 2F9, 2C5, 2H9, 1F11and 2F10 was IgG1 subtype, with a κ light chain. Hybridoma supernatantswere used in all ELISA and Western blot assays described in this sectionunless noted otherwise.

Differential ELISA of MAbs. In an attempt to confirm that the MAbsreacts specifically with the OPN protein but not with the His-tag,GSTOPN (GST cleaved), a recombinant human OPN without His-tag, was usedin addition to h-FL OPN as the test antigens in the ELISA testing.Antibodies 2E11, 1F11, 2C5, 2H9 and 2F10 were tested against h-FL OPNwith 6×His-tag, GSTOPN (recombinant OPN with GST tag cleaved), controlswine viral protein with 21×His-tag, and PBS with 3% none fat dry milk.The figure showed that these five antibodies tested positive againsth-FL His OPN and h-FL OPN with GST cleaved but negative against eitherthe control His-tagged protein or PBS with 3% none fat dry milk(negative control). A control His protein, a recombinant swine viralprotein with 21×His-tag but no OPN sequence, was used alongside as thenegative antigen. FIG. 4 shows that all the MAbs reacted with h-FL OPNand GSTOPN but not with the control His protein confirming that theseantibodies were specifically reactive against OPN.

Cross-species OPN reactivities of MAbs. Although the MAbs were madeagainst recombinant human OPN they might also react with other mammalianOPNs since they were products of B cells from OPN. In order to elucidatethis, the supernatants from these five monoclonal hybridomas were probedagainst h-FL OPN and m-FL OPN in an ELISA (FIG. 5A) and in Western blot(FIGS. 5B & 5C). In the ELISA, the wells were coated with h-FL OPN andm-FL OPN, respectively. The results showed that all five antibodies2E11, 2C5, 2H9, 1F11, and 2F10 recognized OPN from both species. In theWestern blot 20 ng of His-tagged h-FL OPN were loaded in each lane of12.5% polyacrylamide gel. After the electrophoresis the proteins wereblotted onto a PVDF membrane and each lane was cut into a strip to beprobed with each antibody. The result showed that all five MAbs 2E11,2C5, 2H9, 1F11 and 2F10 identified a 66-KDa band. FIG. 5C is arepetition of the Western blot in FIG. 5B with His-tagged m-FL OPN.Similarly, all five antibodies (2E11, 2C5, 2H9, 1F11 and 2F10)identified a 66-KDa band.

The results demonstrated that these antibodies reacted with both humanand mouse full-length OPNs. In ELISA, all five antibodies demonstratedsignificantly higher responses to mouse OPN than the pre-immune sampledid. In addition, their responses to both human and mouse OPNs werecomparable. This result was further corroborated by the Western blotwhere all five antibodies recognized the 66 kDa mouse and human OPNbands. In contrast, the pre-immune sample did not have any positivesignal (FIGS. 5A, B, C).

Antibodies against OPN Fragments. It was shown above that these fivemonoclonal antibodies recognized full-length OPN (65 KDa) (FIGS. 6A, B).In order to further identify their reactivity to the OPN epitopes twoOPN fragments, N-terminal OPN (40 KDa) and C-terminal OPN (25 KDa), weretested against these five monoclonal antibodies in ELISA and in Westernblot. Assays were performed as above. The ELISA test showed that MAbs2C5, 2H9 and 2F10 recognized the N-terminal OPN (FIGS. 6A, B), 1F11recognized the C-terminal OPN (FIGS. 6A, C), while 2E11 recognized C-OPNmore strongly than 2E11. The ELISA results were further confirmed byWestern blot against N-terminal OPN and C-terminal OPN.

Further Antibody Epitope Mapping and Comparison. In an order todetermine whether these five MAbs bind to the same epitopes on OPN, thebiotinylated MAbs 1F11 and 2F10 were used to compete with the fiveunlabeled MAbs for binding to specific OPN epitopes using a competitionELISA assay. Epitope mapping involves a more molecular approach, e.g.,phage display assay or using synthetic peptide. In this assay, thebiotinylated MAbs compete with unlabeled MAbs for binding to specificOPN epitopes. Each microtiter plate well was coated with 20 ng/100 ulhuman FL OPN. In FIG. 7A, each well was incubated with PBS+milk(control) or one of the five unlabeled MAbs followed by addition of 1F11biotinylated antibody. In FIG. 7B, biotinylated 2F10 was used in placeof 1F11-biotinylated antibody. Biotinylated Ab binding was detected byincubation with HRP-conjugated streptavidin, followed by TMB substrate.Both FIGS. 7A and 7B showed that the biotinylated antibodies were onlyinhibited by their unlabeled counterparts, resulting in decreasedabsorbance readings. This showed that 1F11 and 2F10 recognize OPNepitopes different from those of the other MAbs.

Reactivities Against Native OPN. The results above have demonstratedthat the five antibodies reacted with recombinant OPN. In order toconfirm that they also react with native OPN these five MAbs were usedin Western blots to probe proteins from rat skull and kidney tissuelysates (FIG. 8). The analysis was carried out as described in FIG. 5Bexcept the 10-20 μg of lysate proteins samples were loaded in each lane.After electrophoresis and blotting the blot was probed with each MAb anddeveloped with the ECL detection system. The band corresponding tonative OPN from those two tissue lysates were identified with the fiveMAbs, which had similar molecular weights as the positive control(recombinant h-FL OPN of 65 KDa). Similarly, when tissue lysates fromwild type, heterozygous and OPN^(−/−) mice were used in the Western blotthe OPN bands were present only in the wild type and the heterozygoussamples. These results showed that these MAbs interacted with OPN of ratand mouse species.

Immunohistochemistry in Mouse Spinal Discs. The ability of the MAbs tobind native OPN in situ was also investigated in animal tissue withimmunohistochemistry. Since OPN in mouse embryos indicates that the geneis expressed in early stages of bone formation by some preosteoblastsand osteoblasts and in some cells of the marrow (Mark, et al., JHistochem Cytochem, 35:707-716, 1987; Mark, M. P., et al., Cell TissueRes, 251:23-30, 1988), biotinylated 1F11 was used to probe OPN in tissuesections of whole embryos from 15-day old wild-type mice. Mouse embryowas fixed in PLP and cryoprotected in 20% sucrose in PBS and then frozenin OCT compound. The thickness of tissue sections was 5 μm. The antigenswere retrieve using citrate buffer under high heat. The endogenousperoxidase activity was eliminated by 0.3% hydrogen peroxide andnonspecific binding was blocked by 2% goat serum in 1% PBS-BSA. 3.3μg/ml of biotinylated 1F11 was used to probe (FIGS. 9B, 9C) and 1.0μg/ml of purified mouse IgG as a negative control (FIG. 9A). ABC Elitkit and DAB were used as signal detection for test sample slides and1:100 HRP-goat anti-mouse antibody followed with DAB substrate fornegative control. Finally the slides were stained with methylene blue.The low-power view (50×) of embryonic mouse showed strong expression ofOPN (dark brown) in mouse spine (FIG. 9B). High-power view (200×) showedthe expression of OPN was located the bone sections of the spinal dices(FIG. 9C). The mouse IgG, negative control showed nothing in the spinalbone structure (100×; FIG. 9A). The black arrows were pointed to thebone section on the spinal disc. Intense staining was found in thespinal dices but not in other parts of the bone structure and tissues(FIGS. 9 B, C). In contrast, when a negative control pure mouse IgGirrelevant to OPN was used as the probe, there were no signals in thespinal bone area (FIG. 9A). Therefore, our MAbs were able tospecifically detect OPN molecules in situ.

Cells Adhesion to OPN. Liaw, et al., (1994) showed that OPN promotedcell adhesion in a dose-dependent manner. The MAbs specific fordifferent OPN epitopes present ideal tools to study structure andfunction relationship of OPN in cell adhesion, using a cell bindinginhibition assay. In order to determine the optimal dosage of OPN as acell adhesion promoting factor, 2-fold serial dilutions of osteopontinfrom 500 nmol/L to 3.9 nmol/L were used to coat microtiter plate wells.After 30-60 minutes of incubation, the degrees of adhesion to allthree-cell lines, MEF, MCF7, and PyVT, reached plateau with OPN at 62.5nM (as measured by A_(595nm)). Further increases in OPN concentrationdid not improve cell attachment significantly (FIG. 10A). On the otherhand, when OPN concentrations were 7.8 nM or less, there was essentiallyno cell adhesion as shown by insignificant absorbance readings.

After the optimal OPN concentration was determined, the ability of theMAbs to inhibit cell adhesion was tested. FIG. 10B showed that the fiveantibodies (tested at saturation concentrations) decreased cell adhesionby ˜20-50%, with 2C5 having the least inhibitory effect (21% inhibition)and 2F10 having the most (51% inhibition) (FIG. 10B). This result wasconsistent with the observation that 2C5 had the lowest activity inELISA. In contrast, the inhibitory effect was not significant (only 12%inhibition) for 1E3, a control antibody irrelevant to OPN.

Development of Quantitative ELISA for OPN: Optimization of ELISAConditions. MAb 2F10 specific for the N-terminal of OPN and MAb 1F11specific for the C terminal were evaluated as a capture-detectionantibody pair in a sandwich ELISA for OPN quantification. To aiddetection of signals, the detection antibody was biotinylated. In thisassay design 2F10 was designated the capture antibody and biotinylated1F11 was the detection antibody. Hence, this antibody selection ensuredthat only relatively intact OPN molecules be recognized andquantitatively determined.

The chessboard reagent titration experiments (setting up the assay in a“chessboard” pattern takes into account the variability inherent to theassay plate and the detection system) were carried out to define optimalassay parameters. Representative results from multiple experimentsshowed that 2F10 at 1.5 μg/ml and 1F11 at 0.6 μg/ml would be the optimalantibody concentrations for the sandwich ELISA.

Establishment of ELISA for Full-length OPN: Establishment of StandardCurve. There are commercially available kits for the detection ofosteopontin. Immunobiological Diagnostics (Minneapolis, Minn.) sells ahuman osteopontin assay kit that detects full-length osteopontin at asensitivity of 5 ng/ml, Assay Designs (Ann Arbor, Mich.) offers a humanosteopontin enzyme immunometric assay kit with a detection sensitivityof 5 ng/ml, and R&D Systems (Minneapolis, Minn.) has a human osteopontinimmunoassay (Quantikine) with undefined sensitivity. All of these assaysdetect full-length osteopontin protein. As an initial test of ourantibodies and to compare our results to the commercially availableassays, we developed an ELISA for the detection of full-lengthosteopontin using two of our antibodies, 2F10 (N-terminal specific) and1F11 (C-terminal specific). Both antibodies were biotinylated, and usedin turn as either the capture or detection antibody in a sandwich ELISAassay. We found that the combination of these antibodies could detectrecombinant osteopontin efficiently (FIG. 11), with a conservativesensitivity of approximately 0.45 ng/ml. This result suggests that ourassay is more sensitive to low osteopontin levels compared tocommercially available assay kits, and that the sandwich ELISA can bedeveloped using our new monoclonal antibodies. Thus, the sandwich ELISAgenerates a standard curve that can be used to provide accuratedeterminations of OPN concentrations in test samples.

Levels of Plasma OPN in PTC Patients. In attempt to evaluate OPN as abiomarker for cancer, the sandwich ELISA was applied to measure OPNlevels in PTC (papillary thyroid carcinoma) patients. Eighteen healthycontrols were analyzed by antigen-capture ELISA to determine the basalplasma OPN levels. Group 1 consists of 4 PTC patient plasma samples andGroup 2 consists the other 10 PTC patient plasma samples. OPN levelswere higher in both groups of PTC patients (Group 1: mean 16.73 ng/ml,range 0.27-34.59 ng/ml; Group 2: mean 40.56 ng/ml, range 13.06-84.66ng/ml) (FIGS. 12A, B) than in controls (mean 2.8 ng/ml, range 0-16.76ng/ml). The percent difference of Group 1 and Group 2 from controlpopulation was 78.67 and 213.97%, respectively.

OPN Levels in of Plasma OPN in Bodily Fluids of Kidney TransplantPatients. The OPN level from pre- and post-kidney transplant patients'urine and plasma samples were measured in the sandwich ELISA for OPNquantification. Neat samples (plasma or urine) at 50 μl/well were loadedon top of pre-coated capture antibody 2F10 followed by addition ofbiotinylated monoclonal antibody 1F11 (detecting antibody).HRP-conjugated streptavidin and TMB substrate were added to developcolor. FIG. 12A shows the mean concentration for each group was derivedusing the mean A_(405nm) value read against the standard curve in FIG.11. Percent difference=(Sample Mean Concentration−Normal Meanconcentration)/Normal Mean Concentration×100. FIG. 12B shows thedistribution of individual A_(405nm) values for each group. Three datapoint from the control group (A_(405nm)=0.631, 0.608 and 0.506) wereconsidered outliers and removed from the data analysis. All patientsamples were represented here and included in the calculations. Thepost-urine, pre-and post-plasma samples showed higher OPN level thancontrols (mean of post-urine 16.38, range 0-127.84, mean of pre-plasma13.79, range 0.15-33.49, mean of post-plasma 18.16, range 2.24-53.78).The percent differences of kidney transplant patient's samples fromcontrol were 76.72, 62, and 86.79% respectively (FIGS. 12A, B). UrineOPN level from pre-transplant patients samples did not show significantdifferences from the control group. Additionally, post-transplantsamples (plasma and urine) had higher OPN levels than theirpre-transplant counterparts, probably because of the wound resulted fromtransplantation surgery (Liaw, et al., 1998).

1. A method of determining the presence of C-terminal fragments ofosteopontin in a sample suspected of comprising C-terminal fragments ofosteopontin, the method comprising: a) providing i) a sample suspectedof containing C terminal osteopontin fragments from which full lengthosteopontin and fragments of osteopontin comprising N-terminal aminoacid residues of osteopontin have been removed or separated from anyC-terminal fragments present and ii) one or more antibodies specific forepitopes located on the C-terminal of osteopontin selected from thegroup consisting of 2E11 produced by a hybridoma deposited as ATCCAccession No. PTA-11449 and 1F11 produced by a hybridoma deposited asATCC Accession No. PTA-11448; b) determining the presence of C-terminalfragments of osteopontin by determining specific binding of fragments inthe sample with the one or more antibodies of step a).
 2. The method ofclaim 1, wherein said antibodies specific for epitopes located on theC-terminal of osteopontin are specific for epitopes located at aminoacids 167-314 or amino acids 169-314 of the C-terminus of theosteopontin peptide.
 3. The method of claim 1, wherein said method isautomated.
 4. A method of determining the presence of N-terminalfragments of osteopontin in a sample suspected of comprising N-terminalfragments of osteopontin, the method comprising: a) providing i) asample suspected of containing N-terminal osteopontin fragments fromwhich full length osteopontin and fragments of osteopontin comprisingC-terminal amino acid residues of osteopontin have been removed orseparated from any N-terminal amino acid residues present and ii) one ormore antibodies specific for epitopes located on the N-terminal ofosteopontin selected from the group consisting of 2C5 produced by ahybridoma deposited as ATCC Accession No. PTA-11447, 2H9 produced by ahybridoma deposited as ATCC Accession No. PTA-11446 and 2F10 produced bya hybridoma deposited as ATCC Accession No. PTA-11450; b) determiningthe presence of N-terminal fragments of osteopontin by determiningspecific binding of fragments in the sample with the one or moreantibodies of step a).
 5. The method of claim 4, wherein said antibodiesspecific for epitopes located on the N-terminal of osteopontin arespecific for epitopes located at amino acids 1-166 or amino acids 1-168of the N-terminus of the osteopontin peptide.
 6. The method of claim 4,wherein said method is automated.